Modified hepatitis C virus (HCV) Type 5 envelope E2 protein and application thereof
A hepatitis C virus, hepatitis C technology, applied in applications, viral peptides, antiviral agents, etc.
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Embodiment 1
[0048] Example 1: Amplification of the 1-6 genotype HCV envelope E2 protein gene containing HVR1 and deleting HVR1:
[0049] HCV envelope protein E1E2 genes of types 1a, 1b, and 2a were donated by Professor C.M.Rice of Rockefeller University in the United States; HCV envelope protein E1E2 genes of types 3a, 4, 5, and 6 were donated by Professor J.K.Ball of Nottingham University in the United Kingdom. See Table 1 for the gene sequence. Design and synthesize primers according to the corresponding sequences of 7 HCV envelope E2 genes of 1-6 genotypes (1a, 1b, 2a, 3a, 4, 5, 6), and amplify E2 protein by polymerase chain reaction (PCR) Extracellular segment gene (equivalent to 364-661 amino acid residues of HCV polyprotein, 364-383 amino acid residues as secretory signal peptide of E2 protein). The sequences of amplification primers (upstream primer FP1 and downstream primer RP) are shown in Table 2. Reagents for PCR amplification are products of Promega Corporation, USA.
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Embodiment 2
[0058] Embodiment 2: Identification of 1-6 genotype HCV envelope E2 protein expression product:
[0059] Human embryonic kidney (HEK) 293T cells were subcultured in DMEM medium containing 10% fetal bovine serum. When the cells were 80% confluent, the cells were digested with 0.25% trypsin, subcultured and inoculated on 35mm cell culture dishes, and cultured overnight. Combine 4 μg of plasmids constructed above with 7 strains of HCV envelope E2 protein expression plasmids pCI-E2t (including HVR1), pCI-E2tΔ (deleted HVR1) and empty vector pCI-neo with 10 μl of transfection reagent lipid Plastid Lipofectamine (product of Invitrogen, USA) was mixed and transfected into HEK293T cells, and the operation was performed according to the instructions. After adding the plasmid-liposome mixture to the cell culture dish, place the cell culture dish in a cell culture incubator at 37°C, 5% carbon dioxide, and saturated humidity, and add 1ml of complete DMEM culture solution containing 10% fe...
Embodiment 3
[0061] Embodiment 3: Mouse gene immunization and antibody detection:
[0062] Use a plasmid extraction kit (product of Qiagen, Germany) to prepare and purify a large number of HCV envelope E2 protein expression plasmids pCI-E2t, pCI-E2tΔ and empty vector pCI-neo of the above 1-6 genotypes, and dissolve them in PBS buffer (pH 7.4), adjust the concentration to 100μg / ml, immunize Balb / C mice, immunize 10 mice with each plasmid, inject 0.1ml of the plasmid solution into the tibialis anterior muscle on each side, and the equivalent dose is 20μg of the plasmid per mouse. A total of three immunizations were performed with an interval of two weeks. The day before each immunization (0, 2, 4 weeks respectively, the first blood collection time was set as 0 weeks) and every two weeks after the last immunization (7 times in total, the time was 6, 8, 10, 12, 14, 16 and 18 weeks) Blood was collected from the orbit of the mice, and E2t (including HVR1), E2tΔ (deleted HVR1) and HVR1 antibodie...
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